The long-term objective of this application is to develop methods for protection against orally ingested pollutants and toxicants by dietary manipulation of small intestinal cytochromes P4501A. The hypothesis is that induction of small intestinal cytochrome P4501A levels by dietary constituents serves as a major role in protecting the organism from bioactivatable chemical carcinogens and other toxicants. The P4501A(s) protect by facilitating xenobiotic metabolism and excretion to the intestinal lumen, and/or by bioactivating the xenobiotics, which then bind to enterocyte constituents and are cleared with the sloughed-off enterocyte. The following specific aims will test this hypothesis: 1. To determine the basal P450 constituents of intestine of rats fed a diet based on purified components. These studies will provide a basal metabolic background from which to assess effects of induction by dietary constituents on intestinal P4501A metabolism of xenobiotics. 2. To characterize the induction of rat intestinal P4501A forms by beta- naphthoflavone (BNF) as a model for dietary inducers. Studies will include effects of route of BNF administration, BNF does, synergistic enhancement of induction by glucocorticoids, time to achieve induction, duration of induction, situation of induced P4501A along the length of small intestine, situation of induced P4501A in the crypt or villus, relative extents of induction of P4501A and 1A2 and the basis for differences from hepatic induction of these forms. 3. To determine the role of dietary constituents in regulating intestinal P4501A forms. Initially dietary iron, selenium, cruciferous regulating intestinal P4501A forms. Initially dietary iron, selenium, cruciferous vegetables and indole-e-carbinol will be investigated and other examples will be sought. Possible novel mechanisms of induction of intestinal P4501A by dietary constituents will be investigated. Techniques to be applied to the above aims include reverse transcriptase-PCR, immunoblots, Northern blots, immunohistochemistry, and metabolic activity measurements. 4. To determine the capability of small intestinal P4501A to produce presystematic xenobiotic excretion and thus detoxification: a) An ex vivo intestinal preparation will be used to determine the relative extents of enterocyte elimination of xenobiotic or metabolite into the intestinal lumen or systemic circulation. The former case would result in detoxification, the latter case would have potential toxic consequences. b) Radiolabeled xenobiotics will be used to assess the capacity of intestinal P4501A in vivo to detoxify by enhancing excretion as conjugated or free metabolite or metabolite bound to sloughed-off enterocyte. These studies will provide the basis for manipulating the protective role of intestinal P4501A by dietary factors, thus by achieving additional protection against the systemic toxicity of ingested xenobiotics.
